Biochanin obstructs human serum albumin from non-enzymatic glycation: an in vitro approach.

Various serum proteins, like Human Serum Albumin (HSA) and others, are susceptible to glycation and the formation of Advanced Glycation End Products (AGEs). Diabetes and other diseases are associated with AGE development. Recently, isoflavones have been studied for their therapeutic benefits. In the present study, we glycated HSA with Methylglyoxal (MGO) with and without the test compound, i.e., Biochanin A (BCA), to test its antiglycating capacity. We studied the biochemical and biophysical effects of glycation on HSA with and without BCA and also took the help of the in silico technique. Analytical methods included intrinsic and extrinsic fluorescence, polyacrylamide gel electrophoresis (PAGE), UV spectroscopy, far UV circular dichroism, and others. For structural comprehension, TEM and SEM were used. Molecular docking and simulation were employed to observe BCA-HSA's site-specific interaction. Since HSA is a therapeutically relevant protein involved in many disorders, this study's findings are important.Communicated by Ramaswamy H. Sarma.

Biophysical insight into the binding mechanism of epigallocatechin-3-gallate and cholecalciferol to albumin and its preventive effect against AGEs formation: An in vitro and in silico approach.

Advanced glycation end products (AGEs) are produced non-enzymatically through the process of glycation. Increased AGEs production has been linked to several diseases including polycystic ovary syndrome (PCOS). PCOS contributes to the development of secondary comorbidities, such as diabetes, cardiovascular complications, infertility, etc. Consequently, research is going on AGEs-inhibiting phytochemicals for their potential to remediate and impede the progression of hyperglycaemia associated disorders. In this study human serum albumin is used as a model protein, as albumin is predominantly present in follicular fluid. This article focusses on the interaction and antiglycating potential of (-)-Epigallocatechin-3-gallate (EGCG) and vitamin D in combination using various techniques. The formation of the HSA-EGCG and HSA-vitamin D complex was confirmed by UV and fluorescence spectroscopy. Thermodynamic analysis verified the spontaneity of reaction, and presence of hydrogen bonds and van der Waals interactions. FRET confirms high possibility of energy transfer. Cumulative antiglycation resulted in almost 60 % prevention in AGEs formation, decreased alterations at lysine and arginine, and reduced protein carbonylation. Secondary and tertiary structural changes were analysed by circular dichroism, Raman spectroscopy and ANS binding assay. Type and size of aggregates were confirmed by Rayleigh and dynamic light scattering, ThT fluorescence, SEM and SDS-PAGE. Effect on cellular redox status, DNA integrity and cytotoxicity was analysed in lymphocytes using dichlorofluorescein (DCFH-DA), DAPI and MTT assay which depicted an enhancement in antioxidant level by cumulative treatment. These findings indicate that EGCG and vitamin D binds strongly to HSA and have antiglycation ability which enhances upon synergism.

Effect of syringic acid and syringaldehyde on oxidative stress and inflammatory status in peripheral blood mononuclear cells from patients of myocardial infarction.

Oxidative stress and inflammation are considered as therapeutic targets in myocardial injury. The aim of the present study was to investigate the protective effect of syringic acid (SA) and syringaldehyde (SYD) on peripheral blood mononuclear cells (PBMCs) of myocardial infarction (MI) patients. PBMCs from MI patients were cultured in the presence and absence of SA and SYD. The level of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and nitric oxide (NO) was estimated. Reactive oxygen species (ROS) formation, oxidation of lipids, proteins, and activity of antioxidant enzymes were also quantified. To further determine biomolecular changes in treated PBMCs, Fourier transform infrared (FTIR) spectroscopic analysis was done. Molecular docking study was also conducted to evaluate the binding interaction of SA and SYD with various target proteins. SA and SYD treated PBMCs of MI patients showed decreased secretion of TNF-α, IL-6, and NO. Moreover, the content of ROS, level of lipid, and protein oxidation showed diminution by treatment with both the compounds. Enhanced antioxidant defense was also observed in treated PBMCs. The FTIR spectra of treated cells revealed safeguarding effect of SA and SYD on biomolecular structure. The molecular docking analysis displayed significant binding affinity of the two compounds towards TNF-α, IL-6, and antioxidant enzymes. Our findings demonstrated potent antioxidant and anti-inflammatory effects of SA and SYD on PBMCs of MI patients. Thus, SA and SYD supplementation might be beneficial in attenuating oxidative stress and inflammation in MI.

Anti-oxidant and anti-inflammatory effects of cinnamaldehyde and eugenol on mononuclear cells of rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is an autoimmune disorder affecting joints and frequently characterized by initial local and later systemic inflammation. The present study was conducted with the aim to determine the anti-inflammatory and antioxidant effects of cinnamaldehyde and eugenol in the peripheral blood mononuclear cells (PBMC) of RA patients. PBMCs obtained from RA patients were treated with varying concentrations of cinnamaldehyde and eugenol. The levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were monitored in the 24-h culture supernatant of PBMCs. Reactive oxygen species formation, biomolecular oxidation and the activities of antioxidant enzymes were also determined. FTIR analysis was done to determine structural alterations in the PBMCs. Molecular docking was performed to gain an insight into the binding mechanism of eugenol and cinnamaldehyde with pro-inflammatory cytokines. The levels of pro-inflammatory cytokines and markers of oxidative stress were found to be elevated in the PBMC culture of RA patients as compared to the healthy controls. Cinnamaldehyde and eugenol have significantly reduced the levels of cytokines. Reactive oxygen species formation, biomolecular oxidation and antioxidant defense response were also ameliorated by treating PBMCs with both the compounds. FTIR results further confirms cinnamaldehyde and eugenol mediated protection to biomolecules of PBMCs of RA patients. Molecular docking results indicates interaction of cinnamaldehyde and eugenol with key residues of TNF-α and IL-6. Cinnamaldehyde and eugenol were found to exert potent anti-inflammatory and anti-oxidant effects on the PBMC culture of RA patients. So, these compounds may be used as an adjunct in the management of RA.

Post-translational modifications on glycated plasma fibrinogen: A physicochemical insight.

Methylglyoxal (MGO) is a highly reactive α-oxoaldehyde. It reacts with basic amino acids of the proteins to form advanced glycation end products (AGEs). Fibrinogen is a soluble multi-domain glycoprotein whose major function is to form fibrin clots that prevent blood loss upon vascular injury. In the present study, fibrinogen was incubated with varying concentration of MGO for 7 days followed by its biochemical and biophysical analysis. Glycated plasma fibrinogen (MGO-fibrinogen); exhibited hyperchromicity, a drop in tryptophan and intrinsic fluorescence, augmented AGE-specific fluorescence and melting temperature. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed decrease in mobility of MGO-fibrinogen. Structural perturbations in secondary and tertiary structure were identified by fourier transform-infrared spectroscopy (FT-IR), followed by far and near-UV circular dichroism (CD). Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF-TOF) mass spectrometry studies suggested increase in molecular mass of MGO-fibrinogen. Amyloid like aggregates were confirmed by Thioflavin T (ThT), Congo red assay (CR), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The ketoamine moieties, carbonyl content (CO), hydroxymethylfurfural (HMF), superoxide and hydroxyl content were markedly elevated, whereas, total antioxidant capacity (TAC) and free thiol content decreased in MGO-fibrinogen as compared to the native protein. These investigations confirmed the structural and functional alterations in MGO-fibrinogen which leads to different physiological conditions like diabetes mellitus, cardiovascular disease etc.

Elevated DNA Damage, Oxidative Stress, and Impaired Response Defense System Inflicted in Patients With Myocardial Infarction.

BACKGROUND: Ischemic tissue damage in myocardial infarction (MI) is allied with the exaggerated production of reactive oxygen species (ROS) beyond the countering capability of chain-breaking radical scavengers, fallouts in the form of oxidatively burdened myocardial tissue. METHODS: One hundred and twenty five patients with MI were included in the study to evaluate the dynamics of redox status of patients by monitoring the antioxidant potential, biomarkers of oxidative stress, lipid indices, RBC membrane damage when compared to healthy individuals in patients with MI congregated on the basis of Global Registry of Acute Coronary Events (GRACE) score, risk factors, and age. RESULTS: Higher levels of malondialdehyde, 8-hydroxy-2-deoxyguanosine, lipid indices, ROS content, and membrane deterioration in erythrocytes were seen in patients with MI. Furthermore, reduced activities of erythrocyte antioxidant enzymes and lower concentrations of antioxidant molecules, plus reduced total antioxidant capacity, were observed in plasma of all patients with MI with respect to control. However, elevation in oxidative stress was found to be significantly marked in patients having GRACE score >100, risk factors, and MI >45 years when compared to patients with GRACE score ≤100, without risk factors, and MI ≤45 years, respectively. CONCLUSION: These findings indicate the existence of increased oxidative damage and reduced antioxidant potential in patients with MI have a potent relationship with their GRACE risk score, risk factors, and age.

Evaluation of TNF-α and IL-6 Levels in Obese and Non-obese Diabetics: Pre- and Postinsulin Effects.

BACKGROUND: Type 2 diabetes (T2DM) mellitus is a serious implication of obesity. The effect of insulin therapy on levels of inflammatory markers among obese and non-obese diabetics has been inadequately studied. AIM: The study aimed to analyze the preinsulin and postinsulin levels of tumor necrosis factor α (TNF-α) and IL-6 in nonobese and obese T2DM patients. MATERIALS AND METHODS: We assessed TNF-α and IL-6 levels in healthy controls (n=10) and diabetic patients (obese and nonobese; n=20 each) and analyzed the postinsulin effect on TNF-α and IL-6 levels after 24 and 48 weeks. TNF-α and IL-6 levels were also correlated with fasting plasma glucose of obese and nonobese diabetic patients after insulin therapy. RESULTS: There is augmentation of TNF-α and IL-6 levels in diabetic patients and augmentation is more in obese than in nonobese diabetics. The obese group showed a significant decrease (P value<0.05) after 24 weeks of insulin therapy and an extremely significant decrease (P<0.001) in TNF-α and IL-6 levels after 48 weeks of therapy. The nonobese group showed an extremely significant decrease (P<0.001) in TNF-α and IL-6levels after 24 and 48 weeks both. CONCLUSION: There is augmented inflammation in diabetes and it is more in obese diabetics. Insulin therapy tends to counter this inflammation, but the response is delayed in obese diabetics.

Glutathione peroxidase activity in obese and nonobese diabetic patients and role of hyperglycemia in oxidative stress.

BACKGROUND: Both hyperglycemia and obesity are known to cause oxidative stress, which leads to many complications associated with diabetes mellitus. A large number of diabetic patients are obese. Glutathione peroxidase (GPx) is an important indicator of level of oxidative stress. MATERIALS AND METHODS: In the present study, we assessed GPx levels in 20 healthy controls, obese, and nonobese diabetic patients (n=20 each) and analyzed the effect of insulin treatment for 24 and 48 weeks on GPx activity. GPx activity was measured using biochemical method. The GPx activity was also correlated with glycemic status of obese and nonobese diabetic patients [fasting plasma glucose (FPG) levels] after insulin therapy. Statplus software was used for statistical analysis. RESULTS: We found that there is suppression of GPx activity in diabetic patients as compared to healthy controls (70.9 ± 9.6 U/mg protein) and suppression is more in case of obese (23.4 ± 3.8 U/mg protein) than nonobese diabetics (41.5 ± 3.5 U/mg protein). Both obese (26.05 ± 4.03 U/mg protein) and nonobese (48.7 ± 4.8 U/mg protein) diabetics had increase in GPx activity after 24 weeks of insulin treatment. Further, insulin treatment led to improvement in oxidative stress after 48 weeks in both obese (28.4 ± 6.4) as well as nonobese diabetics (51.8 ± 5.4). The nonobese group showed extremely significant (P<0.001) increase in GPx activity after 24 and 48 weeks both, while obese group showed significant (P value<0.05) increase in GPx activity with insulin treatment only after 48 weeks. A negative correlation was found between postinsulin GPx levels and FPG of obese and nonobese diabetics. The correlation was more strong in case of nonobese than obese diabetics. CONCLUSION: Higher levels of oxidative stress in obese diabetics even after control of hyperglycemia by insulin treatment reflect the importance of obesity in contributing to oxidative stress.